One of the major characteristics of the human Red Blood Cell (RBC) is to be the only cell to have a prolonged life span (120 days) despite the absence of a nucleus. The mechanisms of enucleation are suspected (Bessis, 1958; Lichtman, 1981; Qiu et al., 1995), but have not been formally established due to a lack of experimental conditions permitting the massive ex vivo generation of RBCs.
In adult humans, in vivo hematopoiesis results from a dynamic production process situated in the bone marrow, which starts from a minor population of haematopoietic stem cells (HSCs) according to a pyramidal cellular hierarchy (Stem Cell (SC), progenitor and maturation compartments) (Ogawa, 1993) and operates in close contact with the microenvironment (Lemischka, 1997; Friedenstein, 1977; Verfaillie, 1993). In vitro erythropoietic microenvironments were developed in vitro, showing the importance of contact between the hematopoietic and adherent cells (Ohneda et al., 1997; Yanai et al., 1997; Hanspal et al., 1994; Hanspal et al., 1998)). Furthermore erythropoiesis is known to be positively regulated by stem cell factor (SCF), interleukin (IL3), and erythropoietin (EPO) (Zermati et al., 2000; Sato et al., 2000; Dolznig et al., 2002).
If it is apparently easy to obtain almost complete erythroid differentiation (Fibach et al., 1989; Wada et al., 1990; Panzenbock et al., 1998; Freyssinier et al., 1999), the literature data nevertheless show, on reaching the final stage of the different methods of culture proposed, either an important cell proliferation without terminal maturation (Sui et al., 1996; von Lindern et al., 1999), or enucleation in about half of the cells but with a reduced level of amplification (Malik et al., 1998). No set of ex vivo conditions has yet been reported which allows to obtain both a massive proliferation and total enucleation of the erythroblasts.
The expansion of HSCs derived from Cord Blood (CB) in a well-defined stroma-free medium, has been described based on the sequential addition of growth factors (Neildez-Nguyen et al., 2002). Starting from CD34+ cells, this protocol enabled the massive production of pure erythroid precursors that were yet incapable to reach maturation into enucleated cells.
The interest of producing erythrocyte precursors for infusion was reviewed in L. Douay, 2003.